Ratiometric fluorometer

ABSTRACT

A method and apparatus for measuring the concentration of various analytes in a sample using native fluorescence is described. A first light source for producing a first light having a first wavelength is directed at the sample to produce a first emission from the sample, a second light source for producing a second light having a second wavelength is directed at the sample to produce a second emission from the sample. A detecting device for detecting the first and second emissions emitted from the sample, and a controlling device responsive to the detecting device for alternately switching between the first and second light source so that only one light source is directing light at the sample at any one time are employed to excite emissions from the sample to be analyzed. An analyzing device that is responsive to the controlling device for producing a duty ratio is used to determine the analytic concentration of the specific analyte present in the sample.

REFERENCE TO RELATED APPLICATIONS

The instant application claims the benefit of U.S. ProvisionalApplication (Ser. No. 60/167,238) filed on Nov. 24, 1999, incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of contract No.RR-10955 and contract No. RR-14170 awarded by the National Institutes ofHealth (NIH).

FIELD OF THE INVENTION

The present invention relates generally to an analytic apparatus andparticularly to a ratiometric fluorometer for measuring theconcentration of various analytes in a sample.

BACKGROUND

Currently, the predominant technique for measuring dissolved oxygen in asample is a polarographic method involving the monitoring of themodified Clark electrode. This approach involves a relatively expensive,inefficient, and inaccurate method of measuring oxygen concentrations.In particular, the Clark electrode method is adversely affected bysignal drift which in turn affects the long term stability of theelectrode. Furthermore, this method is adversely affected by flow ratesas a result of the electrode consuming amounts of the oxygen to beanalyzed and thus causing the measurements of the oxygen concentrationto be unreliable at best. Finally, while the Clark electrode method is along-standing polarographic approach for measuring dissolved oxygen,practitioners find this method is susceptible to electrical interferencethat adversely affects the accuracy of the oxygen concentrationmeasurements.

Recently, optical methods in measuring the oxygen concentration of asample have been employed in an attempt to overcome the limitations ofthe Clark electrode. These optical techniques attempt to measure theoxygen induced changes in the emission intensity of a sample todetermine the oxygen concentration in that sample. Optical measurementtechniques are based on the premise that long-lived states of manyemissive transition metal complexes (emissive dyes) are quenched atoxygen concentrations of environmental, industrial, and biomedicalinterest. Emissive dyes, once polymer encapsulated or dissolved in themedia being analyzed, can be used to measure oxygen in the gas phase aswell as in an aqueous or biomedia form. These emissive dyes includeseveral tris(diimine)ruthenium (II) complexes and metalloporphyrinsthat, once polymer encapsulated or dissolved in the media underinvestigation, may be used for oxygen measurement. However, systemsemphasizing oxygen induced changes in emission intensity have incurredvarious problems that have resulted in inaccurate measurements of oxygenconcentration of the sample and therefore unreliable results.Measurements obtained by such systems are adversely affected by changesin optical clarity, fluctuations in the source detector, andphotobleaching of the emitter. These non-analyte induced variations inemission intensity of the sample require a relatively expensive andextremely complex system that continuously restandardizes intensitybased sensors in an attempt to obtain accurate measurements.

A second technique used to measure oxygen concentration in a sampleutilizes a frequency modulated excitation to irradiate the sample. Useof a system embodying a frequency modulated excitation apparatus enablesa lifetime dependent phase-shift to be used to measure quenching of along-lived emissive state and thereby obtain the oxygen concentration.Although a frequency modulated phase-based method may be used toeliminate many of the problems associated with emission intensitytechniques, the implementation of such methods based on frequencymodulation requires an extremely expensive and complex system.

A third technique used to measure the oxygen concentration of a sampleutilizes a two-dye method. An optical device is used to measure theemission intensity of both dyes and obtain an intensity ratio used tomeasure the oxygen concentration of the sample. Unfortunately, systemsutilizing the two-dye method are adversely affected by photobleaching ofone or both dyes. This non-analyte induced variation in intensity leadsto gross miscalculations of the intensity ratio and results ininaccurate oxygen concentration measurements.

In addition to oxygen concentration measurements, optical methods havebeen employed to measure pH, Ca²⁺, Mg²⁺, Zn²⁺, heavy metals andtransmembrane potentials of samples which are very important in thebiomedical field. Currently, conventional steady-state methods are usedto determine these types of measurements. However, steady-state systemsembodying optical measurement techniques are prone to errors due tolosses in the optical path, photobleaching, scattering, and backgroundlight. Furthermore, many conventional measurement systems employtechniques that require electrical contact with the object underinvestigation in order for a measurement to be obtained. Frequentrecalibration is needed due to such non-analyte induced variations inemission intensity of the sample. As a result of these problems andlimitations, very strict experimental conditions need to be upheldduring the measurement process resulting in complex measurement systemsand an expensive and inefficient process that yields error-proneresults.

The publication entitled “A Unique Analyzer Combining a Dual EmissionProbe and a Low-Cost Solid State Ratiometric Fluorometer”, with apublication date of September 1999, by Yordan Kostov, Kelly A. VanHouten, Peter Harms, Robert S. Pilato, and Govind Rao, is herebyincorporated by reference. In addition, the publication entitled“Low-cost Device for Ratiometric Fluorescence Measurements”, with apublication date of December 1999, by Yordan Kostov and Govind Rao, ishereby incorporated by reference.

For example, U.S. Pat. No. 3,804,535, issued Apr. 16, 1974 to Rodriguez,the disclosure of which is hereby incorporated by reference, discloses adual wavelength photometer apparatus for measuring an analyte in asample. The measurement of an oxygenation characteristic of a bloodsample is described as a typical use of the apparatus. Light sources aresequentially directed through a sample of blood at a predeterminedrecurring rate and the difference in intensity of the emerging resultantbeams (called the reference light beam and measure light beam) ismeasured. The only light beam affected by the oxygenation of the bloodsample is the measure light beam allowing the oxygen content of theblood sample to be measured. However, measuring an analyte of a sampleby measuring the difference in intensity has proven to be a problematicand unreliable method. As discussed above, such intensity basedmeasurements are adversely affected by changes in optical clarity due tolosses in the optical path between the reference and measurement lightsources and the photometer, photobleaching, scattering and backgroundlight, and fluctuations in the source and detector. Moreover, thesenon-analyte induced variations in intensity make continualrestandardization of the intensity based circuit shown in FIG. 1 of the'535 Patent a requirement.

Such an instrument is described in U.S. Pat. No. 4,803,049 issued toHirschfeld et al., the disclosure of which is hereby incorporated hereinby reference. Patent No. '049 discloses a pH-sensitive optrode (optrode)for monitoring the pH of a sample of physiological fluids, such asblood. An organic dye that fluoresces when excited by a light having aparticular wavelength and whose fluorescence emission intensity varieswith the levels of pH in physiological fluids, such as blood, isutilized to generate a fluorescent signal used to measure the pH of ablood sample. The organic dye molecules are covalently attached to asupport material that in turn is in contact with the blood sample. Whenilluminated, the organic dye disposed on the support is caused tofluoresce. The intensity of the organic dye fluorescence varies with thelevels of pH of the blood sample. However, as discussed above, thisintensity based method of pH measurement is adversely affected bychanges in optical clarity due to losses in the optical path between thesupport material and the photomultiplier tube, scattering and backgroundlight, and fluctuations in the source and detector. Moreover,photobleaching of the dye leads to gross changes in the fluorescenceintensity of the dye resulting in unreliable and inaccurate pHmeasurements.

For the foregoing reasons, there is a need for an apparatus that canaccurately and inexpensively measure the concentrations of oxygen, pH,Ca²⁺, Mg²⁺, Zn²⁺, heavy metals and transmembrane potentials in a sample.

SUMMARY

It is an object of this present invention to provide new and improvedtechniques for measuring various analytes of a sample.

It is yet another object of this invention to provide an analyticapparatus for accurately, non-invasively, quickly and continuouslymeasuring various analytes of a sample.

It is still another object of this invention to provide an analyticapparatus for measuring various analytes of a sample using nativefluorescence.

It is a further object of this invention to provide an analyticapparatus for measuring various analytes of a sample that does notrequire electrical contact with the sample under investigation.

It is still a further object of this invention to provide an analyticapparatus for measuring various analytes of a sample to obtainequilibrium measurements.

It is still a further object of this invention to provide a low-cost,relatively simple analytic apparatus for measuring various analytes of asample that does not require recalibration and constant upgrades inparts and equipment.

It is still yet another object of this invention to provide an analyticapparatus for measuring various analytes of a sample that utilizes aninternal reference that negates non-analyte induced intensity changes inthe sample.

An analytic apparatus is disclosed for determining various analytes of asample according to the teachings of this invention. According to oneembodiment of the invention, the analytic apparatus includes a firstlight source for producing a first light having a first wavelength thatis directed at the sample to produce a first emission from the sample, asecond light source for producing a second light having a secondwavelength that is directed at the sample to produce a second emissionfrom the sample, a detecting device for detecting the first and secondemissions emitted from the sample, a controlling device responsive tothe detecting device for alternately switching between the first andsecond light source so that only one light source is directing light atthe sample at any one time, and an analyzing device that is responsiveto the controlling device for producing a duty ratio which is used todetermine the analytic concentration of the sample.

The foregoing and other objects and advantages will appear from thedescription to follow. In the description, reference is made to theaccompanying drawing which forms a part thereof, and in which is shownby way of illustration specific embodiments for practicing theinvention. These embodiments will be described in sufficient detail toenable those skilled in the art to practice the invention, and it is tobe understood that other embodiments may be utilized and that structuralchanges may be made without departing from the scope of the invention.The following detailed description is therefore, not to be taken in alimiting sense, and the scope of the present invention is best definedby the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings in which:

FIG. 1 is a schematic block diagram of an analytic apparatus accordingto one embodiment of the present invention.

FIG. 2a is a chart illustrating the output of the emission detectoraccording to one embodiment of the present invention.

FIG. 2b is a chart illustrating the output of the amplitude detectoraccording to one embodiment of the present invention.

FIG. 2c is a chart illustrating the output of the voltage analyzeraccording to one embodiment of the present invention.

FIG. 2d is a chart illustrating the output of the hysteresis triggeraccording to one embodiment of the present invention.

FIG. 3 is a second embodiment of the present invention.

DETAILED DESCRIPTION

The present invention is directed to a new apparatus for measuringvarious analytes in a sample. This apparatus is based on the above noteddiscovery that certain luminescent dyes have dual-emittingcharacteristics when dissolved in room temperature solutions or whenpolymer encapsulated, and when excited by modulated light.

The present invention is based on the apparatus and method that enablesvarious analytes in the sample to be measured either by utilizing: (1) adual-excitation probe comprising a dual-excitation indicator in solutionor polymer-encapsulated; or (2) a dual-emission probe comprising adual-emission dye in solution or polymer-encapsulated. The dualexcitation probe posses at least two maximum in its excitation spectrum.The dual emission probe possess at least two peaks in their emissionspectrum. The emission peaks may be both fluorescent emissions orfluorescent and phosphorescent emissions. It should be understood thatwhen the probe is in contact with the analyte, the amplitude of one ofthese peaks is affected—either because of the changes in the electronstructure of the dye or because of dynamic quenching. These fluorescentand phosphorescent emissions are measured in one embodiment of thepresent invention and used to measure the analyte of the sample. In thepresent invention, the duty ratio of the hysteresis trigger 35 (T1/T2)is proportional to the luminescence intensity ratio. The period of thesquare wave (T1+T2)—the illumination cycle time—is proportional to theconcentration of the emissive dye (fluorophor) used in the sample.

As a pH analyzer, the analytic apparatus of the present invention isconfigured to measure fluorescence emissions of indicator solutions madeup of dual-excitation dyes so that the pH concentration of a sample maybe measured. A pH sensitive carboxydichlorofluororescein may be used inthe present invention as a dual excitation indicator to measure the pHlevel of a sample. In a preferred embodiment, a 5-(and6-)-carboxy-2′7′-dichlorofluorescein, mixed isomers (5-(and 6-CDFC)),pKa 3.8, is used as a dual-excitation indicator for measuring the pH ofa sample.

As an oxygen analyzer, the analytic apparatus of the present inventionis configured to measure the relative fluorescence and phosphorescenceintensity of the short-lived singlet and long-lived oxygen-quenchabletriplet of [(dppe)Pt{S₂C₂(CH₂CH₂—N-2-pyridinium)}][BPh₄], where dppe is1,2-bis(diphenylphosphino)ethane, a dual-emission dye. The analyticapparatus may also be configured to measure other dual emission-dyessuch as several carboxy SNAFL-1 and carboxy SNAFL-2 dyes, as well asSNARF-1 and SNARF-2 dyes, when these dual-emitting dyes or molecularprobes are polymer encapsulated, or dissolved in the media beinganalyzed.

The present invention includes a sensing dye that is aheterocyclic-substituted platinum-1,2-enedithiolate,L₂Pt{S₂C₂(Heterocycle)(R)}. This new class of luminescent molecules isboth fluorescent and phosphorescent in room temperature solutions andwhen polymer encapsulated or dissolved in solution. The two emissions,fluorescence and phosphorescence, are assigned to an intraligandcharge-transfer singlet, (¹ILCT*), and triplet, (³ILCT*) withconsiderable 1,2-enedithiolate π to heterocycle π* character. Thequantum yields and lifetimes of these complexes in room temperaturesolutions vary with the heterocycle, the ancillary L₂-group, theR-group, and solvent polarity. As a class of molecules, the solutionlifetimes of the singlet are generally less than 1 ns while the tripletlifetimes vary from 1-16 μs. All of the emissive dye molecules in thisfamily have an excitation maximum near 470 nm, making them compatiblewith the analytic apparatus described herein.

In a preferred embodiment, the[(dppe)Pt{S₂C₂(CH₂CH₂—N-2-pyridinium)}][BPh₄] dye molecule isimmobilized in cellulose acetate (CA) which contains 75% by weighttriethylcitrate (TEC), where the TEC percentage is based upon the weightof CA and is used to plasticize the polymer. The lumiphore loading is 03% of the combined CA/TEC weight. It is well understood by those skilledin the art that the sensitivity of polymer immobilized lumiphores tooxygen quenching is controlled, by a large extent, by the polymer andplasticizer content. In the preferred embodiment, the CA/75% TEC polymeris utilized because of the stability of the polymer when cast intofilms, and the compatibility of the polymer with certain preferredoxygen concentrations and pressures.

Excitation of the [(dppe)Pt{S₂C₂(CH₂CH₂—N-2-pyridinium)}][BPh₄] dyemolecule in CA75% TEC under N₂ results in the dual-emissioncharacteristics of this family of complexes. The singlet and tripletmaximum are at 560 and 675 nm, respectively, and the triplet/singletratio under N₂ is ≈1.03. The selective loss of the ³ILCT* emission inair results in a drop of the triplet/singlet ratio to 0.62. Excitationunder O₂ results in nearly a complete loss of the ³ILCT* emission. Whilethe average ³ILCT* lifetime under N₂ is 14 μs, it decreases to 4.6 μs inair. A phase shift decrease of greater than 20° at 50,000 Hz accompaniesthe decrease in lifetime. Both observations are consistent with oxygenquenching of the ³ILCT*.

Furthermore, the triplet state of these dual-emitting dyes can beselectively quenched by a number of electron, proton, hydrogen atom andenergy transfer processes allowing for the measurement of a range ofanalytes such as pH, Ca²⁺, Mg²⁺, Zn²⁺, heavy metals and/or transmembranepotentials, to include a few.

The relative phosphorescence/fluorescence or fluorescence/fluorescenceor phosphorescence/phosphorescence intensity ratios may be used in thepresent invention to measure a range of analytes present in a sample.The concentration of the analytes to be measured determines the emissionintensities of the fluorophor and thus the luminescence intensity ratio.Therefore, the differences in spectra can be used to measure analytes inany number of ways. In one embodiment of the present invention, theanalytic apparatus measures the ratio of two different samples. Inanother embodiment of the present invention, the analytic apparatusmeasures the ratio of intensity when one sample is excited on twodifferent wavelengths. As still another embodiment of the presentinvention, the analytic apparatus measures the ratio of the emissions ontwo different wavelengths.

Referring now to FIG. 1, a diagram of an analytic apparatus according apreferred embodiment of the present invention is shown. Analyticapparatus 1 includes a first light source 3, a second light source 6, acontroller 30 for controlling the first and second light sources, anemission detector 20 (detector) and an analyte analyzer 40 (analyzer),all interconnected using circuitry commonly known to those skilled inthe art.

The first light source 3 includes a LED 4 which produces a firstexcitation light 11 at a first wavelength. The first light source 3 iscoupled to a first voltage controlled current source 5. Similarly, thesecond light source 6 includes a LED 7 which produces a secondexcitation light 12 at a second wavelength, wherein the second lightsource 6 is coupled to a second voltage. controlled current source 8.The first excitation light 11 has a wavelength configured to excite afluorescence emission 13 in the sample when the sample is irradiated bythe first excitation light 11. The second excitation light 12 has awavelength configured to excite a fluorescence emission 14 in the samplewhen the sample is irradiated by the second excitation light 12.

Emission detector 20 is configured to sequentially detect thefluorescence emission 13 and the fluorescence emission 14 emitted fromthe sample. The emission detector 20 converts the emitted fluorescence13 and the emitted fluorescence 14 into electrical signals correspondingto the luminescence intensity of both fluorescence emissions andtransfers, via an electrical coupling, the converted signals to theanalyzer 40. Analyzer 40 processes the electrical signals and derives aluminescence duty ratio (described below) for at least one cycle offluorescent emissions. Analyzer 40 then compares the calculated ratiowith predetermined values to determine the concentration of the analytein the sample.

As shown in FIG. 1, the emission detector 20 preferably includes atranslucent cuvette or a like container 24 having an internal dimensionfor holding a sample to be illuminated, a first excitation filter 21 forfiltering the excitation light 11 produced by the LED 4, and a secondexcitation filter 22 for filtering the excitation light 12 produced bythe LED 7. It should be understood that the cuvette 24 is described tofacilitate understanding of the present invention, and does not form apart thereof. Any vessel performing the functions described herein maybe used with the analytic apparatus 1 without departing from the scopeof the present invention. A photodetector 25 converts the fluorescenceemissions emitted by the sample to corresponding electrical signals. Anemission filter 23 disposed intermediate the sample and thephotodetector 25 is utilized to monitor the fluorescence emissions. Theelectrical signals corresponding to the fluorescent emissions areamplified by a transimpedance amplifier 26 and passed through aband-pass filter 27 that both (i) removes steady-state components whichoriginate from ambient light that enters the optical path of thefluorescent emissions detected by the photodetector 25, and (ii)diminishes the level of the low and high frequency noise thataccompanies the fluorescent emissions. The outputs of the band-passfilter 27 are shown in FIG. 2a.

With reference to FIG. 1, the analytic apparatus 1 also includes thecontroller 30 that controls the illumination cycle of the analyticapparatus 1 by alternating the first light source 3 and the second lightsource 6 in response to the electrical signals produced by the emissiondetector 20. Controller 30 includes an amplitude detector 33, for takingthe absolute value of the electrical signals produced by the emissiondetector 20 and assigning a sign to the one of the electrical signalscorresponding to one of the fluorescent emissions. The controller 30further includes a voltage analyzer 36 that controls a pair of switchesSW1 and SW2, in response to the electrical signals output by theemission detector 20, that both enable the first light source 3 and thesecond light source 6 to operate in a sequential manner and direct theamplitude detector 33 to assign a negative value to the amplitude of oneof the electrical signals corresponding to one of the fluorescentemissions.

As can be seen from FIG. 1, the amplitude detector 33 includes arectifier 31 and a multiplier 32. The rectifier 31 detects the amplitudeof the electrical signals corresponding to the fluorescent signalsproduced by the emission detector 20. One of the electrical signalscorresponding to the fluorescent emissions is transmitted to themultiplier 32 via the switch SW2 that is activated by the voltageanalyzer 36. In a preferred embodiment, the amplitude of the electricalsignal corresponding to the second fluorescence emission is assigned anegative value by transmitting that signal through the multiplier 32.Multiplier 32, in turn, converts the positive amplitude of the secondfluorescence signal output by the rectifier 31 to a negative amplitudeby multiplying the first fluorescence signal by (−1). Alternatively, theelectrical signal corresponding to the first fluorescence emission maybe assigned a negative amplitude while the amplitude of the electricalsignal corresponding to the second fluorescence emission may remainpositive. The outputs of the amplitude detector are shown in FIG. 2b.

Referring to FIG. 1 again, the voltage analyzer 36 includes anintegrator 34 for integrating the electrical signals corresponding tothe fluorescence emissions output by the amplitude detector 33, and atrigger 35 with hysteresis (hysteresis trigger) for directing theswitches SW1 and SW2 in response to the output of the integrator 34, toenable one of the light sources and enable or disable the multiplier 32,respectively. As a result, the output of the hysteresis trigger 35 is asquare wave, as can be seen in FIG. 2d. The time T1 illustrates the timethat the first light source 3 is on for part of the illumination cycle.The time T2 illustrates the time that the second light source 6 is onduring the remainder of the illumination cycle. Analyzer 40 computes theoutput of the trigger 35 to measure the duty ratio of hysteresis trigger35 and the period of the square wave output by the hysteresis trigger35. Analyzer 40 includes a timer 41 and divider 42 for measuring theduty ratio and the period of the hysteresis trigger 35. The analyzer 40will be described in detail below.

As discussed above, the duty ratio of the hysteresis trigger 35 ismeasured by the analyzer 40 having the timer 41 and divider 42. Thecurrent that flows through the integrator 34 during an illuminationcycle, equal to a half period of the modulating frequency, is given bythe following equations: $\begin{matrix}{{I_{1} = {\int_{0}^{{1/2}\quad f}{{I_{F1} \cdot \sin}\quad 2\quad \pi \quad {{ft}\quad \cdot {t}}}}},\quad {I_{2} = {- {\int_{0}^{{1/2}\quad f}{{I_{F2} \cdot \sin}\quad 2\quad \pi \quad {{ft}\quad \cdot {t}}}}}},\quad {or}} & \left( {1,2} \right) \\{{I_{1} = {\frac{2}{\pi}I_{F1}}},\quad {I_{2} = {{- \frac{2}{\pi}}\quad I_{F2}}},} & \left( {3,4} \right)\end{matrix}$

where f is the modulation frequency of the excitation light, and I_(F1)and I_(F2) are the amplitudes of the fluorescence signals correspondingto each wavelength of integration. The electrical signals produced bythe photodiode 25 are harmonic, as the high frequency components of thelight source modulation are suppressed by the band-pass filter 27. Theoutput voltage of the integrator 34 is proportional to the integral ofthe current flowing through a capacitor contained therein (not shown forclarity). In a preferred embodiment, the initial and final voltages arethe lower U_(lo) and the upper U_(up) threshold of the trigger thusmaking the integration times T₁ and T₂ calculable by the equations$\begin{matrix}{{T_{1} = \frac{{RC} \cdot \left( {U_{up} - U_{lo}} \right)}{n_{1} \cdot I_{1}}},\quad {T_{2} = {\frac{{RC} \cdot \left( {U_{up} - U_{lo}} \right)}{n_{2} \cdot I_{2}}.}}} & \left( {5,6} \right)\end{matrix}$

Here, RC is the integration constant of the integrator 34. In equations3-6, the time intervals are assumed to be equal to a whole number ofhalf waves—as the length of the integration time is usually more than10³ times longer than the length of one halfwave, this does notintroduce significant error. When the ratio of the intensities iscalculated by $\begin{matrix}{{\frac{I_{E1}}{I_{E1}} = {K\frac{T_{2}}{T_{1}}}},} & (7)\end{matrix}$

the ratio of the time intervals (T₂/T₁) is proportional to theluminescence intensity ratio. The period of the square wave (T₁+T₂) isproportional to the concentration of the fluorophor. In a preferredembodiment, the ratio of the time intervals is evaluated using the timer41 (digitizing the lengths of the intervals) and divider 42, in analyzer40, for calculation of the ratio. Alternatively, the ratio of the timeintervals may be evaluated by measurement of the DC component (dutyratio, DR) of the square wave. As DR=T₁/(T₁+T₂), may be calculated bythe equation $\begin{matrix}{\frac{T_{2}}{T_{1}} = {\frac{1}{{DR} - 1}.}} & (8)\end{matrix}$

In accordance with these principles, a detailed description of apreferred embodiment of the analytic apparatus 1 performing an analytemeasurement will now be discussed with reference to FIG. 1 so that onemay better understand the present invention. The first light source 3includes a high-brightness blue LED 4 having a maximum wavelength of 470nm and 40 nm FWHM, and configured to excite a fluorescence emission fromthe sample. The second light source 6 includes a green LED having amaximum wavelength of 520 nm and 40 nm FWHM and configured to excite afluorescence emission from the sample. For example, the blue LED 4, partnumber MBB51TAH-T (Micro Electronics Corp., Santa Clara, Calif.) with aluminous intensity of 4000 mcd, and the green LED 4, part number NSPG500 (Nichia, Mounville, Pa.) with a luminous intensity of 5000 mcd, areused in the preferred embodiment. In the preferred embodiment, thedual-excitation indicator is carboxydichlorofluorescein-pH sensitive insolution.

In alternative embodiments, alternative dual excitation indicator dyesknown in the art may be used to measure the pH in a sample. In suchalternative embodiments, LEDs 4 used to match the excitation maxima ofalternative dual excitation indicators are utilized as the first andsecond light sources to excite the fluorescence emissions from thesample having these aforementioned indicators, without departing fromthe scope of the invention.

It should further be understood that other dual-excitation indicatorsmay be utilized to measure other analyte in a sample. In such cases, thesample may contain dual-excitation dyes that may be used to measure theanalyte and alternative LEDs 4 used to match the excitation maxima ofthese alternative dual-excitation indicators will be utilized as thefirst and second light sources to excite the fluorescence and/orphosphorescence emissions from the sample.

The first light source 3 and the second light source 6 are sequentiallyenabled and disabled by the controller 30 so that the first and secondlight sources work completely out of phase. In other words, when thefirst light source 3 emits modulated light, the second light source 6 isoff and when the second light source 6 is enabled thereby permitting thesecond light source 6 to emit modulated light, the first light source 3is turned off.

The analyte measurement procedure begins with the first light source 3turned on and the switches SW1 and SW2 in position I. The light from thefirst light source 3 passes through a band-pass filter 21 having a470±10 nm bandwidth and excites fluorescence 13 in the sample containedin cuvette 24. The fluorescence emission 13 passes through the emissionfilter 23 having a 590±40 nm bandwidth and is detected by the photodiode25 and amplified by the transimpedance amplifier 26 with an appropriatesecond amplification stage. In the case that other dual-excitationindicators are used to measure the analyte of the sample, the emissionand the excitation filters are configured to the specifications of theexcitation and emission maxima of the indicator used.

In a preferred embodiment, the LEDs 4 and 7 are driven using both afirst voltage controlled current source 5 and a second voltagecontrolled current source 8. The LED current is modulated by a modulatedvoltage source 9 at frequency of 3 kHz. The LEDs are operated in pulsemode at a peak current of 60 mA—twice as much as the nominal rating. Thetotal output optical power from the blue LED 4 is approximately 3.3 mW,and the power from the green LED 7 is approximately 1.4 mW.

In a preferred embodiment, the resulting electrical signal is passedthrough the second order, narrow band-pass active filter 27 with unitygain to remove the steady state components and diminish the low and highfrequency noise. The center frequency of the pass-band is chosen to beequal to the modulation frequency of the LEDs 4 and 7. The output of theband-pass filter 27 is transmitted through the rectifier 31 foramplitude detection.

At this point in the measurement procedure, the switch SW2 is inposition I allowing the output of the rectifier 31 to be transmitted tothe integrator 34 wherein the first fluorescent signal corresponding tothe fluorescent emission 13 is integrated. The output of the integrator34 is connected to the trigger 35 with hysteresis which controls theswitches SW1 and SW2. As the output of the amplitude detector 33 ispositive, the output of the integrator 34 gradually increases, as can beseen in FIG. 2c, towards an upper threshold of the hysteresis trigger35. When this upper threshold is reached, the hysteresis trigger 35changes the positions of switches SW1 and SW2 to position II enablingthe second light source 6 to illuminate the sample with modulated lightand disabling the first light source 3 (turning the first light source 3off).

The light from the second light source 6 passes through the excitationoptical filter 22 and excites a second fluorescent emission 14 in thesample in cuvette 24. The fluorescence emission 14 passes through theemission optical filter 23 and is detected by the photodiode 25 and thenamplified by the transimpedance amplifier 26. The resulting electricalsignal is passed through the band-pass filter 27 to remove the steadystate components and diminish the low and high frequency noise. Theresulting electrical signal is transmitted through the rectifier 31 foramplitude detection.

At this point in the measurement procedure, the switch SW2 is inposition II allowing the output of the rectifier 31 to be transmitted tothe multiplier 32 wherein the amplitude of the electrical signal thatcorresponds to the second fluorescent emission 14 is multiplied by (−1),as can be seen in FIG. 2b. The output of the multiplier 32 is thentransmitted to the integrator 34 wherein the fluorescent signalcorresponding to the fluorescent emission 14 is integrated. As theoutput of the amplitude detector 33 is negative, the output voltage ofthe integrator 34 now gradually decreases, as can be seen in FIG. 2c,towards a lower threshold of the hysteresis trigger 35. When this lowerthreshold of the hysteresis trigger 35 is reached, the latter changesthe switches SW1 and SW2 back to the position I thereby enabling thefirst light source 3 and disabling the multiplier 32. At this point, theillumination cycle is repeated.

As a result, on the output of the trigger 35 appears a square wave, asseen in FIG. 2d. Its duty ratio (T1/T2) is proportional to theluminescence intensity ratio. The period of the square wave (T1+T2) isproportional to the concentration of the fluorophor used. The duty ratiois measured by the analyzer 40 having the timer 41, for measuring thetimes T1 and T2 and the period of the square wave (T1+T2) output by thehysteresis trigger 35, and the divider 42 for producing the duty ratio.

Referring now to FIG. 3, a diagram of an analytic apparatus according toa second preferred embodiment of the present invention is shown.Analytic apparatus 101 includes a first light source 103, a controller130, an emission detector 120, and an analyte analyzer 140 (analyzer)all interconnected using circuitry commonly known to those skilled inthe art.

The light source 103 includes a LED 104 which produces excitation light107 at a specific wavelength configured to excite fluorescence 109 andphosphorescence 110 from the dual-emission probe present within thesample disposed within a cuvette 124. LED 104 is coupled to a modulatedvoltage source 106 through a current source 105.

Emission detector 120 is configured to sequentially detect thefluorescence and phosphorescence emissions emitted from the sample andconvert the aforementioned emissions into electrical signalscorresponding to the luminescence intensity of the emissions. Ananalyzer 140 processes the electrical signals corresponding to thefluorescence and phosphorescence emissions and derives afluorescence/phosphorescence duty ratio (described below) for at leastone emission cycle of fluorescence and phosphorescence emissions.Analyzer 140 compares the calculated duty ratio with predeterminedvalues to detect the concentration of the analyte in the sample.

As shown in FIG. 3, emission detector 120 (detector) preferably includesa translucent cuvette or a like container 124 having an internaldimension for holding a sample to be illuminated, and an excitationfilter 121 for filtering the excitation light 107 produced by LED 104.It should be understood that the cuvette 24 is described to facilitateunderstanding the present invention, and does not form a part thereof.Any vessel performing the functions described herein may be used withthe analytic apparatus 101 without departing from the scope of thepresent invention. A first photodetector 125 and a second photodetector127 respectively convert the fluorescence emission 109 and thephosphorescence emission 110, emitted by the sample, to correspondingelectrical signals. A first emission filter 122 disposed intermediatethe sample and the first photodetector 125 is utilized to monitor thefluorescence emission 109. Similarly, a second emission filter 123disposed intermediate the sample and the second photodetector 127 isutilized to monitor the phosphorescence emission 110.

With reference to FIG. 3, the electrical signals corresponding to thefluorescence and phosphorescence emissions 109 and 110 produced by thefirst and second photodetectors 125 and 127 are amplified bytransimpedance amplifiers 126 and 128, respectively. Emission detector120 further includes a band-pass filter 129 that both (i) removessteady-state components which originate from ambient light that entersthe optical path of the fluorescent and phosphorescent emissionsdetected by the photodetectors 125 and 127, and (ii) diminishes thelevel of the low and high frequency noise, that accompanies thefluorescent and phosphorescent emissions, from the electrical signalsproduced by the photodetectors.

As can be seen from FIG. 3, the analytic apparatus 101 also includes acontroller 130 that controls a detection cycle of the analytic apparatus101 by sequentially enabling the first photodetector 125 and the secondphotodetector 127 in response to the electrical signals produced by thedetector 120. Controller 130 further includes an amplitude detector 133that takes the absolute value of the electrical signals produced by theemission detector 120 and assigns a sign to one of the electricalsignals corresponding to the fluorescent 109 or phosphorescent 110emissions. The controller further includes a voltage analyzer 136 thatcontrols a pair of switches SW1 a and SW2 a, in response to theelectrical signals output by the emission detector 120, that both enablethe first photodetector 125 and the second photodetector 127 to operatein a sequential manner and direct the amplitude detector 133 to assign anegative value to the amplitude of one of the electrical signalscorresponding to one of the fluorescent and phosphorescent emissions,respectively.

As can be seen from FIG. 3, the amplitude detector 133 includes arectifier 132 and a multiplier 134. The rectifier 132 detects theamplitude of the electrical signals corresponding to the fluorescent andphosphorescent signals produced by the emission detector 120. One of theelectrical signals corresponding to the fluorescent and phosphorescentemissions is transmitted to the multiplier 134 via the switch SW2 a thatis activated by the voltage analyzer 136. In a preferred embodiment, theamplitude of the electrical signal corresponding to the phosphorescenceemission is assigned a negative value by transmitting that signalthrough the multiplier 134. Multiplier 134, in turn, converts thepositive amplitude of the phosphorescence signal output by the rectifier132 to a negative amplitude by multiplying the phosphorescence signal by(−1). Alternatively, the electrical signal corresponding to thefluorescence emission may be assigned a negative amplitude while theamplitude of the electrical signal corresponding to the phosphorescencesignal may remain positive. The output of the amplitude detector isshown in FIG. 2b.

Referring to FIG. 3 again, voltage analyzer 136 includes an integrator135 for integrating the electrical signals corresponding to thefluorescence and phosphorescence emissions output by the amplitudedetector 133, and a hysteresis trigger 137 for directing the switchesSW1 a and SW2 a in response to the output of the integrator 135, toenable one of the photodetectors and enable or disable the multiplier134, respectively. As a result, the output of the hysteresis trigger 137is a square wave, as can be seen in FIG. 2d. The time T1 illustrates thetime that the first photodetector 125 is on during the emission cycle.The time T2 illustrates the time that the second photodetector 127 is onduring the emission cycle. Analyzer 140 computes the output of thehysteresis trigger 137 to measure the hysteresis trigger duty ratio.Analyzer 140 includes a timer 141 and divider 142 for measuring thehysteresis trigger duty ratio, as described above.

In accordance with these principles, a detailed description of apreferred embodiment of the analytic apparatus 101 performing an analytemeasurement will now be discussed with reference to FIG. 3 so that onemay better understand the present invention. The light source 103includes a blue LED 104, part number MBB51TAH-T (Microelectronics,Santa, Clara, Calif.) having a maximum wavelength of 470 nm is used.LEDs that cover part of the light spectrum needed to excite theparticular dual emissive dye may be utilized.

The sample containing the analyte to be measured is placed in a cuvette124 equipped with a screw top, a septum, and inlet and outlet lines (notshown) to allow gas flow. In a preferred embodiment,[(1,2-bis(diphenylphosphino)ethane)Pt{S₂C₂(CH₂CH₂—N-2-pyridinium)}][BPh₄]—oxygensensitive in film—is used as the dual-emissive dye. This luminescent dyeis immobilized at 0.3% by weight in CA/TEC and cast in a 0.5 mm thickfilm. The film is trimmed and mounted to a quartz cuvette insert (notshown) with Super 77 spray adhesive (3M, Saint Paul, Minn.) and disposedinside the cuvette 124. In alternative embodiments, alternative dualemission indicator dyes may be used to measure the oxygen in the sample.In such alternative embodiments, the appropriate LED used to excitefluorescence and/or phosphorescence from the particular dye are used asthe light source. In such alternative embodiments, other analyte in thesample may be measured by utilizing dual emission dyes that aresensitive to the analyte to be measured. In these alternativeembodiments, the appropriate LED may be used to excite fluorescence andphosphorescence or two different fluorescence from the sample in themanner described above, without departing from the scope of theinvention.

The modulated light emitted from the LED (f_(mod)=1.5 kHZ) is directedto the sample through a 470±30 nm band-pass filter mounted on thecuvette wall. The excitation light emitted by the blue LED 104 causesthe dual-emissive probe to sequentially emit fluorescence andphosphorescence due to the singlet and triplet emissions (discussedabove). The fluorescence and phosphorescence emissions are monitoredthrough 570±40 nm and 680±22 nm band-pass filters, respectively. Thecorresponding light intensities are detected by two large active-area(13 mm²) PIN-photodiode detectors 125 and 127. The electrical signals,corresponding to the fluorescence and phosphorescence, are amplified andpassed through a band-pass filter 129 to both (i) reject steady-statecomponents, which originate from ambient light entering the optical pathbetween the sample and the photodetectors, and (ii) diminish the levelsof low and high frequency noise. After amplitude detection and signassignment, the signals are serially fed through the switch SW2 a to theintegrator.

When SW1 a and SW2 a are in position I, the fluorescent signal isintegrated. As it is positive, the output voltage of the integrator 135gradually increases to the upper threshold of the hysteresis trigger137. This switches SW1 a and SW2 a switch to position 11 where thephosphorescent signal is integrated. As it is negative (discussedabove), the integrator's 135 output voltage now gradually decreases.When the output voltage reaches the lower threshold of the hysteresistrigger 137, SW1 a and SW2 a switch back to position I and the processis repeated.

The above-described techniques may also be applied to the measurement ofvarious other kinds of analyte in a sample. In addition, it is to beunderstood that various other types of measurements may be achieved withthe present invention. Such measurements include the measurement of theratio of two different samples.

It should be understood that other dual-excitation probes anddual-emission probes, besides the several described above, may also beappropriate for measuring the oxygen, pH, Ca²⁺, Mg²⁺, Zn²⁺, heavy metalsand/or transmembrane potentials concentration of a sample.

It will therefore be seen that the foregoing represents a highlyadvantageous approach to analyte measurement in a sample. The terms andexpressions employed herein are used as terms of description and not oflimitation, and there is no intention, in the use of such terms andexpressions, of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.

For example, alternative light sources may be used to illuminate thesample such as laser diodes, lasers, incandescent lamps, andsemiconductor light sources (to name a few) without departing from thenovel spirit and scope of the present invention. Moreover, excitationlight from the ultraviolet to the visible range may be used to irradiatethe sample and the apparatus of an alternative embodiment may beconfigured to analyze emissions from the visible to the infrared rangeto determine the concentration of analytes present in a sample withoutdeparting from the novel spirit and scope of the present invention.

We claim:
 1. An apparatus for measuring an analytic concentration of asample comprising: a sample containing a dual wavelength-absorbingluminescent compound or a dual wavelength-emitting luminescent compound,a first light source producing a first light having a first wavelengthto be directed at the sample to produce a first emission from thesample; a second light source producing a second light having a secondwavelength to be directed at the sample to produce a second emissionfrom the sample; a detector for detecting said first emission and saidsecond emission emitted from the sample; a controller coupled to saidfirst light source, said second light source and said detector foralternately switching between said first light source and said secondlight source so that only one light source is directing light at thesample; and an analyzer coupled to said controller, wherein saidanalyzer produces a duty ratio which is used to determine the analyticconcentration of the sample.
 2. The apparatus of claim 1, wherein saidfirst light source comprises a first LED and said second light sourcecomprises a second LED.
 3. The apparatus of claim 1, wherein saidcontroller comprises a rectifier for rectifying said electrical signalsproduced by said detector and a multiplier for multiplying theelectrical signals corresponding to one of either said first emission orsaid second emission.
 4. The apparatus of claim 3, wherein saidcontroller means further comprises a first switch and a second switch,an integrator for integrating said electrical signals output by saiddetector and a hysteresis trigger to direct both said first switch andsaid second switch to synchronously switch between a plurality of modes,switching from a first mode to a second mode in response to anintegrator output reaching a first level and switching from a secondmode to a first mode in response to said integrator output reaching asecond level.
 5. The apparatus of claim 1, wherein said detectorcomprises a first excitation filter for filtering said first light, asecond excitation filter for filtering said second light, aphotodetector for converting said first emission and said secondemission emitted by the sample to corresponding electrical signals, anemission filter disposed intermediate the sample and said photodetectorfor monitoring said first emission and said second emission, and aband-pass filter for removing steady-state components and noise fromsaid electrical signals produced by said photodetector.
 6. The apparatusof claim 1, wherein said analyzer comprises a timer for measuring a time1 and a time 2 to produce a square wave period equal to the sum of saidtime 1 and said time 2, which is proportional to a concentration offluorophor used in the sample.
 7. The apparatus of claim 6, wherein saidanalyzer comprises a divider for dividing said time 1 by said time 2 toproduce said duty ratio, which is proportional to a luminescenceintensity ratio of said first emission and said second emission.
 8. Theapparatus of claim 1, wherein the sample comprises a dual-emissive probethat emits said first emission when illuminated by said first light andsaid second emission when illuminated by said second light, said firstemission is fluorescence and said second emission is fluorescence. 9.The apparatus of claim 1, wherein the sample comprises a dual-emissiveprobe that emits said first emission when illuminated by said firstlight and said second emission when illuminated by said second light,said first emission is phosphorescence and said second emission isphosphorescence.
 10. An apparatus for measuring an analyticconcentration of a sample comprising: a sample containing a dualwavelength-absorbing luminescent compound or a dual wavelength-emittingluminescent compound, a light source producing a light to be directed atthe sample to produce a first emission and a second emission from thesample; a detector for detecting said first emission and said secondemission emitted from the sample; a controller coupled to said lightsource and said detector, wherein said controller operates a firstswitch and a second switch in response to said detector; and an analyzercoupled to said controller for producing a duty ratio which is used todetermine the analytic concentration of the sample.
 11. The apparatus ofclaim 10, wherein said light source comprises a LED.
 12. The apparatusof claim 10, wherein said detector comprises a first photodetector forproducing electrical signals proportional to the intensity of said firstemission and a second photodetector for producing electrical signalsproportional to the intensity of said second emission.
 13. The apparatusof claim 12, wherein said controller comprises a rectifier forrectifying said electrical signals produced by said first and secondphotodetectors and a multiplier for multiplying said electrical signalscorresponding to one of either said first emission or said secondemission.
 14. The apparatus of claim 13, wherein said controller furthercomprises an integrator for integrating said electrical signals outputby said detector, and a hysteresis trigger for directing both said firstswitch and said second switch to synchronously switch between aplurality of modes.
 15. The apparatus of claim 12, wherein said detectorfurther comprises an excitation filter for filtering said light, a firstemission filter disposed intermediate the sample and said firstphotodetector for monitoring said first emission, a second emissionfilter disposed intermediate the sample and said second photodetectorfor monitoring said second emission, and a band-pass filter for removingsteady-state components and noise from said electrical signals producedby said first and second photodetectors.
 16. The apparatus of claim 10,wherein said analyzer comprises a timer for measuring a time 1 and atime 2 to produce a square wave period equal to the sum of said time 1and said time 2, which is proportional to a concentration of fluorophorused in the sample.
 17. The apparatus of claim 16, wherein said analyzercomprises a divider for dividing said time 1 by said time 2 to producesaid duty ratio, which is proportional to a luminescence intensity ratioof said first emission and said second emission.
 18. The apparatus ofclaim 10, wherein the sample comprises a dual-emissive probe that emitsfluorescence and phosphorescence when illuminated by said light.
 19. Theapparatus of claim 10, wherein the sample comprises a dual-emissiveprobe that emits fluorescence and fluorescence when illuminated by saidlight.
 20. The apparatus of claim 10, wherein the sample comprises adual-emissive probe that emits phosphorescence and phosphorescence whenilluminated by said light.
 21. A method for measuring an analyticconcentration of a sample, comprising the steps of: directing a firstlight source having a first wavelength at a sample containing a dualwavelength-absorbing luminescent compound or a dual wavelength-emittingluminescent compound to generate a first emission from the sample;directing a second light source having a second wavelength at the sampleto a second emission from the sample; alternatively switching betweenthe first light source and the second light source so that only onelight source is directing light at the sample; detecting the firstemission and the second emission emitted from the sample; and generatinga duty ratio from the first emission and the second emission todetermine the analytic concentration of the sample.
 22. The method ofclaim 21, further comprising the step of generating electrical signalsproportional to the intensity of the first emission and the secondemission.
 23. The method of claim 21, wherein the step of switchingbetween the first light source and the second light source comprises thestep of disabling one light source while enabling the other light sourceto operate the lights in a sequential manner.
 24. The method of claim22, wherein the step of alternatively switching comprises the steps ofrectifying the electrical signals corresponding to one of either thefirst emission or the second emission, and rectifying and multiplying bya constant the electrical signals corresponding to the other of eitherthe first emission or the second emission.
 25. The method of claim 24,wherein the step of alternatively switching further comprises the stepsof integrating the electrical signals corresponding to the firstemission and the second emission, and switching between the first lightsource and the second light source in response to the integratedelectrical signals reaching a first level and a second level.
 26. Themethod of claim 25, wherein the generating step comprises measuring atime 1 and time 2 wherein the time 1 represents a time that the firstlight source is on for part of an illumination cycle and the time 2represents a time that the second light source is on for the remainderof the illumination cycle.
 27. The method of claim 26, wherein thegenerating step further comprises adding the time 1 to the time 2 todetermine a concentration of fluorophor used in the sample and dividingthe time 1 by the time 2 to produce the duty ratio.